Nickel

Example 3

Recombinant Protein Purification

FIG. 5 shows the steps of one of the purifications carried out on the chimera. In the case of GRNLY, this process was shown in an earlier paper [Ibáñez, R., University of Zaragoza. 2015]. It can be seen in FIG. 5A that the P. Pastoris supernatant obtained after induction (lane 1) contains rather diluted proteins. After concentrating same with Pellicom, protein bands are not seen in the permeate (lane 3), but proteins that are much more concentrated than in the supernatant are seen in the concentrate (lane 2). After dialysis (lane 4), the band profile remains similar to the concentrate. Furthermore, protein bands are not seen in the buffer in which the dialysis bag (lane 5) was introduced. Upon addition of the nickel resin, the chimera binds to said resin as it has a histidine tag. After adding the resin (lane 6), the intensity of a band corresponding to a protein of about 40 kDa decreases with respect to the concentrate and dialysate. This band may correspond to the chimera. The fact that this band does not altogether disappear may indicate that the nickel resin was saturated. In the washes performed on the resin, particularly in the first wash (lane 7), it can be seen how the residues of other proteins are removed. Finally, after the elution of the nickel column, a major protein with a molecular weight of about 40 kDa corresponding to the molecular weight of the chimera (lane 11) is clearly observed. As shown in FIG. 5B, it was confirmed by means of immunoblot that this band of about 40 kDa corresponds to the chimera (lane 11). It is also confirmed that the resin was saturated because a band appears in the post-resin dialysis phase (lane 6).

FIG. 6 shows different elution fractions and the pooling of all of them with the exception of elution fraction 1. FIG. 6A shows several bands in the different elution fractions and in the total eluate. The band with the highest intensity has a molecular weight corresponding to the chimera. Furthermore, other bands having intermediate molecular weights are observed, which means that the chimera undergoes partial proteolysis. The band with the second highest intensity has a molecular weight of about 10 kDa, which corresponds to 9-kDa GRNLY, as its molecular weight increases since it is bound to a histidine tag. In FIG. 6B, it was confirmed by means of immunoblot that these bands of about 40 and 10 kDa correspond to the chimeric recombinant protein and to recombinant GRNLY, respectively.

Once the chimera is generated, its functionality must be assured, that is, on one hand the scFv still recognizes the CEA antigen, and on the other hand GRNLY is still cytotoxic.

Example 3

• • (1) Prepared a nickel oxalate dihydrate NiC₂O₄·2H₂O solution A with a concentration of 3 mol/L. Specifically, NiC₂O₄·2H₂O was added to 50 mL of deionized water and stirred for 30 minutes to form a uniformly mixed solution A;
  • (2) Put the solution A into a polytetrafluoroethylene lined autoclave, the volume filling ratio was maintained at 50%;
  • (3) Took a 50 mL beaker, and completely immersed the foamed copper with a length of 7 cm and a width of 1 cm into acetone, 3 mol/L HCl solution, deionized water, and absolute ethanol in sequence, and carried out ultrasonic treatment separately for 30 minutes. Put the processed foamed copper into a polytetrafluoroethylene reactor containing the solution A; put the sealed reactor into a homogeneous hydrothermal reactor, the temperature parameter was set to 180° C., and the reaction time was 18 hours;
  • (4) After the reaction was completed and cooled to room temperature, the foamed copper after the reaction was taken out and washed with absolute ethanol and deionized water for 3 times;
  • (5) Prepared a solution B of tungsten hexachloride WCl₆ with a concentration of 4 mol/L. Specifically, added WCl₆ to 60 mL of deionized water and stirred it for 30 minutes to form a uniformly mixed solution B;
  • (6) Immersed the NiOOH/Cu₂O-grown foamed copper in a polytetrafluoroethylene lined autoclave containing the solution B and sealed it, and the volume filling ratio was maintained at 60%. Put the sealed autoclave into a homogeneous hydrothermal reactor, the temperature parameter was set to 140° C., and the reaction time was 30 hours;
  • (7) After the reaction was completed, cooled to room temperature, took out the foamed copper after the reaction, and washed with absolute ethanol and deionized water 3 times. Put it into a 60° C. vacuum oven or a freeze-drying oven to dry for 6 hours to obtain a NiOOH/Cu₂O/WO₃/CF self-supporting electrocatalytic material. The total loading of NiOOH/Cu₂O/WO₃ was 3 mg/cm². The molar ratio of WO₃, Cu₂O, and NiOOH was 1:0.6:0.05.

Example 4

3D design software and 3D drawing software were used to construct a 3D cylinder model with a diameter of 40 mm and a height of 15 mm, which was converted into an STL file and imported into SLM building software. The model was auto-sliced by the software and imported into an SLM printing system. After heating the substrate to 150° C., the René 104 nickel-based superalloy powder was added to a powder supply tank and then laid. Argon was introduced into the working chamber until the oxygen content was less than 0.1%. Then the printing procedure was carried out, and the steps of laying the powder and scanning the powder by laser were repeated until the printing was completed to obtain a cylinder.

The René 104 nickel-based superalloy powder has a particle size of 15-53 μm, a D₁₀ of 17.5 μm, a D₅₀ of 29.3 μm, and a D₉₀ of 46.9 μm.

The process parameters for SLM are as follows: a laser power of 250 W, a spot diameter of 0.12 mm, a scanning speed of 500 mm/s, a scanning pitch of 0.12 mm, and a thickness of the laid powder layer being 0.03 mm.

The scanning strategy for SLM is a stripe scanning strategy. In the stripe scanning strategy, a layer-by-layer scanning method from bottom to top is adopted, the laser scanning direction is rotated by 67° between adjacent layers, the stripe width is 5 mm, and the overlap between stripes is 0.10 mm. (no contour+solid scanning method is adopted)

The stress relief annealing parameters are as follows: a temperature of 420° C. held for 90 min, and cooling within the furnace.

The SPS parameters are as follows: a graphite mold with a diameter of 40 mm, a heating rate of 60° C./min, a cooling rate of 60° C./min, a sintering pressure of 45 MPa, and a sintering temperature of 1020° C. held for 15 min.

Before and after post-treatments of the fabricated parts, the densities are 98.34% and 99.02%, respectively, and the mechanical properties at room temperature are 987 MPa and 1065 MPa.